Transmembrane proteins (TMPs) play important physiological roles, are the targets of most clinically useful drugs, and constitute 20-30% of most genomes, however they remain poorly characterized at the structural level. High-resolution structures are available for only ten unrelated eukaryotic TMPs (excluding mitochondrial and chloroplast proteins.) Two major barriers to determining structures of eukaryotic TMPs are the difficulty of achieving high levels of expression of TMPs in native conformations and the apparent instability of TMPs leading to heterogeneity and loss of native conformations during expression, purification, and crystallization for x-ray diffraction. This application describes a set of genetic procedures for overcoming these barriers focusing on yeast as a genetically tractable organism that is the expression host with the most successful history of production of eukaryotic TMPs for x-ray crystallography. The overall approach will be to use flow cytometry in conjunction with fluorescent ligands that bind specifically to native conformations of TMPs to screen large libraries of mutated and otherwise modified yeast cells to identify alterations that enhance the levels of expression and stabilities of TMPs. These procedures will initially be applied to G protein Coupled Receptors (GPCRs), including mammalian GPCRs that can be expressed in yeast and the endogenous yeast receptor for -mating pheromone. Both the procedures and the high-expressing strains developed by the project are expected to be applicable to other classes of TMPs. Screening based on binding of fluorescent ligands will be used to: 1) Identify alterations in yeast host strains leading to increased expression of functional receptors. Some of these alterations will be introduced using a new approach, global transcription machinery engineering, that provides the ability to alter expression of multiple genes at the same time. Other alterations will be effected using strains derived from yeast genomic deletion collection and by transforming receptor-expressing strains with libraries of overexpressed yeast genes and mammalian cDNAs; 2) Identify mutations in the genes encoding GPCRs that lead to increased expression; and 3) Identify mutations that render GPCRs resistant to thermal denaturation. As high-level expression of stabilized GPCRs expressed in yeast is achieved, the receptors will be purified, tested for stability in detergent solutions, and subjected to crystallization trials for structure determination.